Dissertations / Theses on the topic 'Thalassemia'

Homozygous α0-thalassemia is a serious autosomal recessive disorder with poor fetal outcome and severe maternal complications. Conventionally, prenatal diagnosis is performed by an invasive test. A non-invasive approach using serial ultrasonography can effectively reduce the need for invasive tests in unaffected pregnancies. For two-dimensional ultrasound prediction, a total of 777 at-risk fetuses were studied from 12 to 20 weeks between 1995 and 2006. At 12–15 weeks’ gestation, the highest sensitivity (98.3%) was achieved by the combination of fetal cardiothoracic ratio (CTR) and/or middle cerebral artery peak systolic velocity (MCA-PSV) at a false-positive rate of 15.8%. At 16–20 weeks’ gestation, the sensitivity of CTR was 100.0%, but the false-positive rate was 5.2%. In contrast, the false-positive rate of MCA-PSV alone was 1.4% and that of the combination of CTR and MCA-PSV was 0%, although their sensitivities were less than 65%. In a cross-sectional retrospective study of 546 samples at-risk and control (268 fetal and 278 neonatal cord blood), the degree of anemia was only mild in 27.5% of the affected fetuses (see chapter 3 for definition of mild anemia). Because MCA-PSV is not very predictive of mild anemia, this may be one of the reasons why MCA-PSV is not very sensitive in predicting an affected pregnancy. A total of 832 at-risk pregnancies were studied using same noninvasive approach at Maternal and Neonatal Hospital of Guangzhou (MNH) and Tsan Yuk Hospital (TYH). The overall sensitivity and specificity of the noninvasive approach was 100% and 95.6% respectively. At MNH, the need for an invasive test was reduced by 78.6%, and all the affected pregnancies were diagnosed before 24 weeks’ gestation. After adequate training and monitoring the quality of the subsequent ultrasound examinations, the results achieved at MNH were comparable to TYH, with at-risk pregnancies including the affected ones being seen at a more advanced gestation at MNH. In a retrospective review of 361 women at risk of carrying an affected fetus, 311 (86.2%) opted for the non-invasive approach using CTR and/or placenta. The cost saving of this non-invasive approach was relatively small (HK$ 2,651) in comparison to the cost of the whole prenatal screening program. On the other hand, the non-invasive approach was more expensive than the direct invasive approach for low MCV couples, as well as couples discordant for α-thalassemia and β-thalassemia. ages. These results support the adoption of non-invasive approach in which routine invasive test or karyotyping is no longer performed. A total of 106 at-risk pregnancies and normal controls were prospectively studied using three-dimensional ultrasonography. Placental volume (PV) at 11-14 weeks, and PV/CRL quotient at 9-14 weeks’ gestation of affected pregnancies were significantly greater than unaffected pregnancies (P<0.05). Using a cut-off point of 1.2ml/mm for PV/CRL quotient to predict an affected pregnancy, the sensitivity, and specificity was 96.2%, and 100.0% respectively.
published_or_final_version
Obstetrics and Gynaecology
Master
Doctor of Medicine

Mestrado em Biologia Molecular e Celular
As mutações nonsense são mutações pontuais que originam codões de terminação prematura (PTCs). A expressão de genes portadores de PTCs pode levar à síntese de proteínas truncadas. As proteínas truncadas caracterizam-se por serem menores e, na maioria das vezes, não possuem função biológica, apesar de poderem ter funções deletérias para a célula. Em condições normais, transcritos portadores de PTCs são degradados rapidamente através do processo de nonsense mediated mRNA decay (NMD). Quando um PTC atinge o sítio A ribossomal, os fatores de terminação da tradução ligam-se ao mesmo e a tradução termina imediatamente. A terapia de supressão consiste numa abordagem terapêutica que tem o objetivo de utilizar compostos de baixo peso molecular para induzir a incorporação de aminoacil-tRNAs quase cognatos, moléculas que possuem complementaridade para dois dos três nucleótidos de um códão de stop, quando o ribossoma atinge um PTC. Assim, a tradução não termina prematuramente. Estudos anteriores mostraram que alguns aminoglicósidos possuem a capacidade de suprimir PTCs responsáveis por doenças, como fibrose quística e distrofia muscular de Duchenne. Algumas mutações nonsense são responsáveis pela β-talassemia. Neste estudo foram utilizados dois aminoglicósidos, canamicina e gentamicina, de modo a avaliar a sua capacidade em aumentar a competitividade de tRNAs quase cognatos com os fatores de terminação da tradução pelo sítio A ribossomal, na presença de um PTC, evitando dessa forma a terminação prematura da tradução.
Nonsense mutations are point mutations that originate premature termination codons (PTCs). The expression of PTC-containing genes may lead to the synthesis of truncated proteins. Truncated proteins are shorter proteins that at most times do not have biological function, but may have deleterious functions for the cell. In regular conditions, PTC-containing transcripts are taken to rapid decay, through nonsense mediated mRNA decay (NMD). When a PTC reaches the ribosomal A-site, translation release factors bind it and translation immediately stops. Suppression therapy is a therapeutic approach that aims to suppress PTCs by using low molecular weight compounds to induce the incorporation of near cognate aminoacyl tRNAs, molecules that show complementarity to two of the three nucleotides of a stop codon, when the ribosome reaches a PTC. Thus, translation does not prematurely terminates. Previous studies have shown that some aminoglycosides have the ability to suppress PTCs responsible for diseases like cystic fibrosis and Duchenne muscular dystrophy. Some nonsense mutations are responsible for β-thalassemia disease. In this study two aminoglycoside compounds, kanamycin and gentamicin, were used in order to evaluate their capacity to increase the competition of near cognate aminoacyl tRNAs with translation release factors by the ribosomal A-site, when the ribosome reaches a PTC, therefore avoiding the premature termination of translation.

α-thalassemia is an inherited globin gene disorder commonly found among the Chinese population. It is composed of both non-deletional and deletional α-globin gene mutations. Classical α-thalassemia presents with red cell microcytosis but silent cases with a normal mean corpuscular volume (MCV) are also seen. Routine laboratory testing methods for large-scale detection of silent α-thalassemia mutations are onerous and time-consuming. Furthermore, methods such as denaturing high performance liquid chromatography (HPLC) or denaturing gradient gel electrophoresis (DGGE) for scanning of point mutations are costly and they require post-PCR separation. High resolution melting (HRM) analysis is an economical, sensitive, and fast method for large scale point mutation scanning. Contamination is significantly reduced with HRM because the process is performed in a closed-tube environment and does not require post-PCR manipulation. We used HRM and multiplex gap-PCR analysis to determine the prevalence of silent α-thalassemia carriers in Hong Kong. Of the 223 hematologically normal blood samples scanned by Roche LightCycler 480®, HRM did not show any sample with a non-deletional α-globin gene mutation of clinical significance. α-multiplex gap-PCR analysis revealed 36 samples (16.1%) with single α-globin gene deletions. The detection of single α-globin gene deletions in samples with a MCV greater than 80 fL indicates that the previously reported prevalence of α-thalassemia mutations in our Chinese population based on MCV screening is under-estimated. The data also suggest that non-deletional α-thalassemia mutations presenting with a normal MCV are very rare, and they most likely present with microcytosis. The fact that most silent α-thalassemia mutations are due to large deletions supports the use of traditional molecular techniques such as gap-PCR for their detection. HRM can be used as an adjunct tool for large-scale population screening of non-deletional mutations. This study provides more accurate data on the prevalence of silent α-thalassemia carriers in the Hong Kong Chinese population. The information will facilitate genetic counseling and risk assessment in families carrying α-thalassemia mutations.
published_or_final_version
Pathology
Master
Master of Medical Sciences

A study on thalassemia intermedia and major patients in Jakarta was initiated to obtain a comprehensive picture of metabolic dysregulation, iron overload, oxidative stress, and cell damage. Data were collected from a group of 10 transfusion-dependent patients in an age range of 11-25 years and another group of 5 frequently transfused (for at least 15 years) patients aged 17-30 years. A third group comprises 5 patients (aged 7 to 14 years) who had not yet obtained transfusions. The 10 controls were voluntary students without diagnosis or clinical signs of thalassemia up to 30 years of age. The study was approved by the Ethical Clearance Board of the Medical Faculty and all blood samples from controls and patients were obtained on fully informed consent. Levels of antioxidants (vitamins A, C, E and B-carotene) and reactive thiols are considerably decreased in transfused patients, whereas signs of iron overload and cell damage are increased (serum iron, ferritin, transferrin saturation, SGOT, SGPT, y-GT, bilirubin).

Background: Nonsense mutations, giving rise to UAA, UGA, UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of about 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy, thalassemia. Currently, there are two approaches to directly overcome diseases caused by nonsense mutations: gene therapy, meaning introduction of an exogenous gene, and translational read-through induced by aminoglycosides, which decrease the accuracy of translation elongation and reduce the efficacy of the translation termination machinery. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. On the other hand, in recent years, many investigators have been studying the development of safe lentiviral vectors to avoid the side effects as activation of oncogenes and transcription silencing. Aim: Our purpose was to verify the effects and clinical utility in b0- thalassemia of translational read-through and gene therapy. Methods: First, we started the development of a cellular model of the b039- thalassemia mutation that could be used for the screening of high numbers of aminoglycosides and analogous molecules. We produced two lentiviral vectors containing the bwt- or b039-thalassemia globin gene under a minimal LCR control region and used such constructs for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different copynumber of the integrated constructs. These clones were then treated with geneticin (G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. Secondly, we screened several thalassemia patients and isolated erythroid progenitors from such individuals resulted homozygous for the b039 mutation. Then, we treated the cells with geneticin and analysed the production of b-globin and adult haemoglobin (HbA) by FACS and HPLC analysis, respectively. Finally, we subjected the b039 erythroid precursor cells to gene therapy, through the use of the lentiviral vector expressing the βwt-globin mRNA, with the aim to verify the increase of HbA through HPLC analysis. Moreover, by treating the transduced cells with mithramycin we wanted to verify the combined effects of gene therapy and fetal haemoglobin (HbF) induction. Main results: We obtained and characterized six clones carrying the b039- globin gene and seven clones carrying the bwt-globin gene, to be used as a control. We then chose two clones, a bwt and a b039, showing similar content of mRNA and used them to test various aminoglycosides to verify their read-through activity. Geneticin (G418) and gentamicin showed the highest capacity to readthrough of nonsense mutation, although the effect of G418 was found to be higher than that displayed by gentamicin. A significant increase in β-globin containing cells was also detected when b039 progenitor cells were treated with G418. This last result was confirmed by HPLC analysis, which showed an increase in the relative concentration of HbA compared to total Hbs. Finally, we obtained a high increase in the proportion of HbA when the same progenitor cells were subjected to gene therapy, through transduction with the lentiviral vector containing the bwt-globin gene, and a clear increment of HbF was detected after treatment with mithramycin. Conclusion: Both the therapeutic approaches for the cure of β0-thalassemias analysed in this work, namely translational read-through and gene therapy, while still presenting many disadvantages, including toxicity of aminoglycosides and the uncertainty of the effects of lentiviral vectors on endogenous genes, can be considered techniques with a high curative potential. Naturally, in the case of translational read-through is hoped the identification of new compounds with a therapeutic effect similar to but greater than that of aminoglycosides, with less toxicity and oral bioavailability, while in the case of gene therapy, further investigations should be made to eliminate its well known disadvantages, primarily including the possible activation of oncogenes.

INTRODUZIONE Le β-talassemie sono una delle malattie genetiche più frequenti in tutto il mondo con 270 milioni di portatori e 350.000 nuovi nati affetti all’anno. Questa malattia è geneticamente caratterizzata dalla perdita di produzione della catena β globinica dell'emoglobina adulta, dovuta a diverse mutazioni nel gene della β-globina. Poiché il gene beta è espresso su entrambi i cromosomi 11, possiamo avere due differenti tipi (e con differente gravità) di beta talassemia a seconda dell’assenza di entrambi o di un solo gene della beta globina: nel primo caso si ha la β talassemia MAJOR o trasfusione dipendente, nel secondo caso si ha la β talassemia MINOR o INTERMEDIA trasfusione indipendente. I nostri studi si concentrano su quest’ultima. L'assenza della catena β globina comporta diverse conseguenze per l'organismo come: - Eritropoiesi inefficace - Il sovraccarico di ferro - Il danno ossidativo Finora sono stati condotti molti studi in diversi campi (genomico, proteomico e nel metabolismo ferro) per garantire una maggiore comprensione di questa malattia. Recentemente si è scoperta una nuova proteina che potrebbe essere un eventuale regolatore o responsabile del sovraccarico di ferro nella β - talassemia; questa molecola è la ferroportina. La Ferroportina (FPN) è l'unico esportatore di ferro finora conosciuto. Essa è espressa in diversi tipi di cellule, tra cui gli enterociti duodenali, gli epatociti, i macrofagi e gli eritroblasti. Pochi anni fa, è stata segnalata l'esistenza di due trascritti alternativi della FPN con o senza le Iron Responsive Elements (IRE) sul loro promotore (FPN1A e FPN1B rispettivamente). L'espressione delle diverse isoforme della ferroportina nonché i meccanismi che la regolano nelle cellule eritroidi della β talassemia non-trasfusione dipendente (NTDT) non sono ancora noti. SCOPO Studiare il profilo di espressione delle due isoforme della ferroportina durante il differenziamento eritroide in colture controllo e di NTDT e chiarire i meccanismi che regolano la loro espressione. MATERIALI E METODI Per questi studi è stato usato un modello di eritropoiesi in vitro derivato da cellule CD34+ provenienti da sangue periferico di volontari sani (controllo) e pazienti NTDT. Il profilo dell'espressione genica delle due isoforme (FPN1A e FPN1B) è stato valutato allo stadio basale (giorno 0) e al giorno 7 e 14 della cultura (stadio di pro eritroblasti e di eritrociti ortocromatici rispettivamente) mediante la tecnica di real-time PCR (2-dCt). La percentuale relativa di ogni isoforma è stata calcolata sulla base dell’espressione della ferroportina totale (FPN1A + FPN1B). La concentrazione di ferro intra and extracellulare è stata analizzata utilizzando un kit di Ferro Assay (Biovision). In esperimenti indipendenti, colture di controllo e NTDT sono state trattate con: ferro (Ferro Ammonio Citrato [FAC] 100μM), Desferal (DFO, 4μM), protoporfirina (SNPP IX 50-20μM), eme (Emina 20-10μM) o perossido di idrogeno (H2O2 0,1mM) per indagare su un possibile ruolo di questi composti nella regolazione della ferroportina. L’espressione della FPN è stata valutata al 14esimo giorno in condizioni standard e nei trattati mediante la tecnica di real-time PCR (2-ddCt; cellule non trattate utilizzate come calibratore). RISULTATI L'espressione ferroportina aumenta durante il differenziamento eritroide, raggiungendo il livello massimo di espressione allo stadio di eritroblasti (giorno 14 di coltura) sia nel controllo sia negli NTDT. La FPN1A è l'isoforma più espressa in entrambe le condizioni. La sua espressione è più elevata negli stadi iniziali e finali dell’eritropoiesi (giorno 0 e 14), mentre l'espressione della FPN1B è maggiore nella fase intermedia di differenziamento eritroide (giorno 7). Degno di nota, l'espressione della FPN1B, anche se inferiore rispetto alla 1A, è significativamente maggiore nelle culture NTDT rispetto ai controlli, in particolare al giorno 14. La concentrazione di ferro intracellulare è diminuita in modo significativo durante il differenziamento eritroide (dal giorno 7 al giorno 14), sia nei controlli sia negli NTDT, tuttavia, al giorno 7 (stadio di eritroblasti) i livelli di ferro nelle culture NTDT sono notevolmente inferiori rispetto ai controlli. L'aggiunta di FAC, DFO, SnPP IX ed Emina nei controlli e nelle colture di NTDT non ha modificato l'espressione della ferroportina rispetto ai non trattati. L’H2O2 aggiunto ai controlli aumenta l'espressione di entrambe le isoforme della ferroportina (FPN1A: cellule non trattate: 1; H2O2: 1.33 FPN1B: cellule non trattate: 1; H2O2: 2.04). I livelli di ferro intra ed extracellulari riflettono i risultati genetici: c'è stato un aumento di ferro extracellulare causa di un aumento di espressione FPN. CONCLUSIONI L'espressione della ferroportina aumenta durante il differenziamento eritroide sia nei controlli sia nelle culture NTDT, suggerendo il suo ruolo nell’esportare il ferro intracellulare in eccesso. In entrambe le condizioni, la FPN1A è l'isoforma più espressa. Tuttavia, l'espressione dell’isoforma 1B non responsiva al ferro, anche se minore rispetto a FPN1A, è significativamente maggiore nei NTDT rispetto ai CTRL. In colture di controllo, l’espressione della FPN, ed in particolare dell’isoforma 1B, sembra essere regolata dall’aggiunta di H2O2. Questi dati suggeriscono che lo stress ossidativo, particolarmente elevato nelle NTDT, potrebbe essere uno dei principali regolatori dell’espressione dell’isoforma 1B, generando così un’importante esportazione di ferro dalle cellule NTDT.
INTRODUCTION β-Thalassemias are one of the most frequent genetic disorders worldwide with 270 million of carriers and 350.000 affected new-borns per year. This disease is genetically characterized by the loss of production of the β globin chain of the adult haemoglobin, due to several mutation within the beta globin gene. Since the beta gene is expressed on both the chromosomes 11, we can have two different type (and severity) of beta thalassemia depending on the absence of both or just one beta gene: in the first case we have the β thalassemia MAJOR transfusion dependent, in the second case we have the β thalassemia MINOR or INTERMEDIA, transfusion independent. Our studies are focused on the last one. The absence of the β globin chain implies different consequences for the organism like as: - Ineffective erythropoiesis - Iron overload - Oxidative damage Many studies have been conducted so far in different fields (genomic, protein expression and regulation, iron metabolism) in order to guarantee a major comprehension of this disease. Recently a new protein came out as a possible regulator/responsible for the iron overload in β thalassemia; this molecule is the FERROPORTIN. Ferroportin (FPN) is the only know iron exporter protein. It is expressed in different cell types including duodenal enterocytes, hepatocytes, macrophages and erythroblast cells. Few years ago it has been reported the existence of two alternative transcripts of FPN with or without an iron – responsive element (IRE) on their promoter (FPN1A and FPN1B respectively). The expression of the different ferroportin isoforms as well as the mechanisms regulating their expression in erythroid cells in non-transfusion dependent β thalassemia syndromes (NTDT) are not known yet. AIM To investigate the expression profile of ferroportin isoforms during erythroid differentiation in control and NTDT cell cultures and to elucidate the mechanisms regulating their expression. MATERIALS AND METHODS An in vitro model of erythropoiesis derived from human peripheral CD34+ cells from healthy volunteers (control) and NTDT patients was used. The expression profiling of FPN isoforms (FPN1A and FPN1B) was evaluated at baseline (day 0) and at day 7 and 14 of culture (pro erythroblasts and orthochromatic erythroblasts stage respectively) by real−time PCR (2−dCt). The relative percentage of each isoform was calculated based on total ferroportin expression (FPN1A+FPN1B). The intracellular iron concentration was analyzed by using an Iron Assay Kit (Biovision). In independent experiments, control and NTDT cultures were treated with iron (Ferric Ammonium Citrate [FAC] 100µM), Desferal (DFO, 4µM), protoporfirin (SnPP IX 50-20µM), heme (Hemin 20-10µM) or hydrogen peroxide (H2O2 0.1mM) to investigate a possible role of these compounds in ferroportin regulation; FPN expression was evaluated at day 14 in standard and treated conditions by real−time PCR (2−ddCt; untreated cells used as calibrator). RESULTS The ferroportin expression increased during erythroid differentiation; with the highest level at the end of erythroblasts stage (day 14 of cultures) both in control and NTDT cultures. The FPN1A was the more expressed isoform in both conditions. Its expression was higher at the initial and final steps of erythropoiesis (day 0 and 14), while FPN1B expression was higher at the intermediate erythroblast stages (day 7). Noteworthy, the FPN1B expression, although lower compared to FPN1A, was significantly higher in NTDT cultures than in control ones, particularly at day 14. The intracellular iron concentration decreased significantly during erythroid differentiation (from day 7 to day 14) both in control and NTDT cultures, however, at day 7 (early erythroblasts stage) the iron levels in NTDT cultures were notably lower than in controls. The addition of FAC, DFO, SnPP IX and Hemin in control and NTDT cultures did not modify the ferroportin expression compared to untreated. H2O2 added to control cells increased the expression of both ferroportin isoforms (FPN1A: untreated cells: 1; H2O2: 1.33. FPN1B: untreated cells: 1; H2O2: 2.04). The intra and extracellular iron levels reflected the genetic results: there was an increase of extracellular iron due to an increase of FPN expression. CONCLUSIONS The ferroportin expression increases during erythroid differentiation either in control than in NTDT cultures, suggesting its role in exporting the excess intracellular iron. In both conditions, the FPN1A is the more expressed isoform. However, the expression of the non−iron responsive FPN1B isoform, although lower compared to FPN1A, is significantly higher in NTDT than in control conditions. In control cultures, FPN expression, and particularly the FPN1B isoform, seems to be up regulated by H2O2 addition. These data suggest that the oxidative stress, notably higher in NTDT conditions, could be one of the major regulator of FPN1B expression, with a major iron export from NTDT erythroblast cells.

Thalassemia is a blood disorder requiring lifelong transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction. We hypothesized that removing this proximate iron compartment as a primary treatment using novel iron chelators, could prevent hastened red cell removal and clinically alleviate the need for transfusion. Novel, highly cell permeable iron chelators, pyridoxal isonicotinoyl hydrazone (PIH) and pyridoxal ortho-chlorobenzoyl hydrazone (o-108) were compared to the present mainstay, desferrioxamine (DFO) and deferiprone (L1), in vitro and in vivo . Treatment of human model beta-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron and reduced oxidative stress as indicated by a decrease in methemoglobin levels. When administered to beta-thalassemic mice, iron chelators mobilized erythrocyte membrane iron, reduced cellular oxidation, and prolonged erythrocyte survival. Consistently, these mice showed improved hematological abnormalities. A beneficial effect as early as the erythroid precursor stage was also determined by normalized proportions of mature versus immature reticulocytes. Remarkably, all four chelators reduced iron accumulation in target organs. Most importantly, o-108 revealed superior activity, decreasing iron in liver and spleen by ~5-fold and ~2-fold, respectively, compared to DFO. Our study demonstrates that iron chelators ameliorate thalassemia in a human and murine model, and validates their primary use as an alternative to transfusion therapy.

Mitochondrial activity and metabolism significantly control hematopoietic stem cell (HSC) function and fate. HSCs change the metabolic state in response to stress signals, such as reactive oxygen species (ROS), which drive HSC entry into cell cycle accompanied by increased mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. However, excessive accumulation of ROS results in oxidative damage of cellular organelles, including mitochondria. Iron is one of the sources of ROS and HSCs can uptake iron but little is known about the effects of iron on HSC metabolism. Recently, we demonstrated an impaired function of HSCs in β-Thalassemia (BThal), a condition of systemic iron overload (IO). We also observed that IO reduces the hematopoietic supportive capacity of BThal BM mesenchymal stromal cells. However, there is no evidence of the direct effect of IO on HSCs in BThal. We hypothesized that IO and the resulting oxidative stress could alter HSC metabolism and function. We found a positive enrichment of iron homeostasis genes in HSCs from thalassemic th3 mice, suggesting increased iron uptake and storage. Consistently, we detected high levels of free reactive iron in the cytoplasm and in mitochondria of th3 HSCs, correlating with high ROS levels. As a result, mitochondria are impaired, with low mass and activity. Interestingly, th3 multipotent progenitors inherited dysfunctional mitochondria since the rescue of mitochondrial activity occurred in the transition to more committed progenitors. In line with mitochondrial dysfunction, th3 HSCs had reduced OXPHOS-derived ATP and relied on glycolysis. In vivo reduction of mitochondrial ROS rescued mitochondrial activity and metabolism, and increased th3 HSC frequency and quiescence, thus indicating that oxidative stress is the cause of mitochondrial dysfunction and potentially HSC defects. Importantly, in vivo administration of iron dextran to wt mice generated intracellular IO and mitochondrial oxidative stress and decreased mitochondrial activity in HSCs, indicating that IO alone is sufficient to impair mitochondria. Our study unveils that IO directly impacts on HSC metabolism by inducing oxidative stress and mitochondrial dysfunction. Alterations in mitochondrial activity and metabolic profile, in response to IO, are expected to alter HSC function. This research will add novel insight about the role of iron in regulating HSC metabolism and provide clues for improving clinical conditions associated to IO, such as BThal.
L'attività e il metabolismo mitocondriali controllano in modo significativo la funzione e il destino delle cellule staminali ematopoietiche (HSC). Le HSC modificano lo stato metabolico in risposta a segnali di stress, come le specie reattive dell'ossigeno (ROS), che guidano l'ingresso delle HSC nel ciclo cellulare accompagnato da un aumento della fosforilazione ossidativa mitocondriale (OXPHOS) e della glicolisi. Tuttavia, l'eccessivo accumulo di ROS provoca il danno ossidativo degli organelli cellulari, compresi i mitocondri. Il ferro è una delle fonti di ROS e le HSC possono assorbire il ferro, ma si sa poco sugli effetti del ferro sul metabolismo delle HSC. Recentemente, abbiamo dimostrato una funzione alterata delle HSC nella β-talassemia (BThal), una condizione di sovraccarico sistemico di ferro (IO). Abbiamo anche osservato che l'eccesso di ferro riduce la capacità di supporto ematopoietica delle cellule stromali mesenchimali talassemiche. Tuttavia, non ci sono prove dell'effetto diretto del sovraccarico di ferro sulle HSC in BThal. Abbiamo ipotizzato che il sovraccarico di ferro e il conseguente stress ossidativo alterino il metabolismo e la funzione delle HSC. Abbiamo trovato un arricchimento positivo dei geni dell'omeostasi del ferro nelle HSC dei topi talassemici th3, suggerendo un aumento dell'assorbimento e dell'immagazzinamento del ferro. Coerentemente, abbiamo rilevato alti livelli di ferro reattivo libero nel citoplasma e nei mitocondri di th3 HSC, che correlano con alti livelli di ROS. Di conseguenza, i mitocondri sono alterati, con ridotta massa e attività. I progenitori multipotenti th3 hanno ereditato mitocondri disfunzionali poiché la correzione dell'attività mitocondriale si è verificata nella transizione verso progenitori più differenziati. In linea con la disfunzione mitocondriale, le HSC th3 hanno una ridotta produzione di ATP mediante OXPHOS e dipendono dalla glicolisi. La riduzione in vivo dei ROS mitocondriali ha ripristinato l'attività e il metabolismo mitocondriali e ha aumentato la frequenza e la quiescenza delle HSC th3, dimostrando così che lo stress ossidativo è la causa della disfunzione mitocondriale e dei potenziali difetti delle HSC. È importante sottolineare che la somministrazione in vivo di ferro destrano a topi wt ha generato eccesso di ferro intracellulare e stress ossidativo mitocondriale e una ridotta attività mitocondriale nelle HSC, indicando che il sovraccarico di ferro da solo è sufficiente per compromettere i mitocondri. Il nostro studio rivela che il sovraccarico di ferro ha un impatto diretto sul metabolismo delle HSC inducendo stress ossidativo e disfunzione mitocondriale. Le alterazioni dell'attività mitocondriale e del profilo metabolico, in risposta al sovraccarico di ferro, potrebbero alterare la funzione delle HSC. Questa ricerca aggiungerà nuove informazioni sul ruolo del ferro nella regolazione del metabolismo delle HSC e fornirà nuove conoscenze utili per migliorare le condizioni cliniche caratterizzate da sovraccarico di ferro, come BThal.

The research described in the present PhD Thesis has been conducted in the context of a multicenter FP7 European Project called THALAMOSS (THalassemia MOdular Stratification System), having as major objective the identification of molecular markers for the development of personalized therapies for hemoglobinopathies, in particular ß-thalassemia. The ß-thalassemias are an autosomal recessive genetic disorders caused by the absence or reduction of ß-globin chains of adult hemoglobin, for which targeted and definitive treatments are, at present, not available. In order to sustain project based on stratification of ß-thalassemia patients according to clinical, genetic and molecular features, we established a systematic collection of cellular samples, the cellular Biobank, containing hematopoietic stem cells isolated from the peripheral blood, expanded, freezed and cryopreserved. To this aim, 75 subjects comprising ß-thalassemia patients and healthy donors have been recruited up to now. They were all characterized for the genotype (in order to detect pathogenic mutations) and possible fetal hemoglobin (HbF)-associated polymorphisms. More importantly, new protocols to efficiently isolate, culture, freeze and thaw hematopoietic stem cells were developed. In particular, we demonstrated that freezing, cryopreservation and thawing steps do not affect the erythroid differentiation potential of the cells and the natural erythroid differentiation process, in terms of kinetics and types of hemoglobin produced by the cells of the same patient. Moreover, we found that the cells stored in the Biobank are responsive, once thawed and sub-cultured, to treatments with known HbF inducers, including mithramycin, resveratrol, butyric acid and hydroxyurea, and are therefore suitable for the identification and development of new HbF inducers to be used for experimental therapeutic strategy for ß-thalassemia. In this context, we demonstrated that the induction effects depend on the subject’s genotype, strongly suggesting that this approach could be very useful to develop personalized therapies. In conclusion, this research activity will allow patients stratification taking into account all the phenotypic/genotypic characteristics of the single individual, in association with in vitro HbF induction under treatment with effective inducers, providing an important opportunity for the research and development of novel therapeutic strategies for ß-thalassemia.

Le emoglobinopatie sono difetti genetici ereditari che originano dal malfunzionamento della proteina adibita al trasporto dell’ossigeno, cioè l’emoglobina (Hb). Tra le emoglobinopatie, l’anemia falciforme (SCD) e la β-talassemia, malattie genetiche che coinvolgono il gene βglobinico, hanno il maggior impatto sulla mortalità e sullo stato patologico e affliggono milioni di persone nel mondo (Weatherall, 2010). Nella β-talassemia, il difetto a carico del gene β-globinico causa uno squilibrio tra le catene α- e β-globiniche nei globuli rossi (RBCs). Questa condizione porta al danno delle membrane cellulari dei globuli rossi, morte precoce ed eritropoiesi inefficace. L’anemia falciforme è dovuta ad una mutazione a cui consegue la sostituzione del sesto amminoacido della β-globina adulta. L’emoglobina che porta questa mutazione (HbS) polimerizza all’interno dei globuli rossi deformandoli e rendendoli più rigidi; questi globuli rossi a falce bloccano il flusso sanguigno nella microcircolazione compromettendo il rilascio dell’ossigeno nei tessuti. Le terapie correnti di queste patologie, oltre alle trasfusioni e alla somministrazione di ferro chelanti, prevedono il trattamento con Idrossiurea (HU), l’unico induttore di emoglobina fetale approvato dalla U.S. Food and Drug Administration (FDA). Ciononostante, la terapia con Idrossiurea raggiunge livelli di emoglobina fetale sufficienti solo nella metà dei pazienti (Steinberg et al., 1997) ed spesso vengono riportati effetti collaterali tra i quali la neutropenia e la leucopenia. Perciò, risulta necessario identificare nuovi induttori di emoglobina fetale per sviluppare una terapia personalizzata per i pazienti affetti da β-talassemia ed anemia falciforme. Complicazioni cliniche severe della β-talassemia e dell’anemia falciforme possono presentarsi rispettivamente in seguito all’accumulo di catene libere α-globiniche o alla produzione di catene β-globiniche difettose. Perciò abbiamo sperimentato la riduzione di emoglobine come potenziale pathway da colpire per sviluppare nuove terapie. La parte I di questa tesi è focalizzata sulla caratterizzazione di nuovi induttori di emoglobina fetale: 1) raccogliendo 33 campioni di sangue da diversi pazienti affetti da β-talassemia abbiamo dimostrato la capacità della Rapamicina di aumentare la produzione di emoglobina fetale, anche in cellule resistenti al trattamento con Idrossiurea; 2) durante la somministrazione di un integratore alimentare a base di Resveratrolo a pazienti β-talassemici abbiamo analizzato l’induzione del messaggero γ-globinico e la produzione di emoglobina fetale nei precursori eritroidi isolati dal sangue periferico; 3) abbiamo testato nuovi composti psoralenici valutando il grado di differenziamento eritroide in cellule eritroleucemiche umane K562 e osservato gli effetti sull’espressione dei geni globinici e sulla produzione di emoglobina fetale nei precursori eritroidi. La parte II di questa tesi è volta a dimostrare l’applicazione terapeutica degli acido peptido nucleici (PNA) nelle cellule eritroidi: 1) abbiamo ottenuto un rilascio efficiente e veloce dei PNA nelle cellule eritroleucemiche umane mediante la coniugazione con una formulazione liposomica, senza evidenti effetti sulla proliferazione (Avitabile et al. 2015); 2) un PNA antisenso diretto contro il messaggero β-globinico è risultato capace di inibire la produzione di emoglobina nelle cellule eritroleucemiche murine (Montagner et al., 2015); 3) un PNA diretto contro la β-globina mutata è stato testato nei precursori eritroidi isolati da pazienti affetti da anemia falciforme.
Hemoglobinopathies are genetic inherited defects that originate from the lack or malfunction of the hemoglobin (Hb) protein. Sickle cell disease (SCD) and β-thalassemia, both prototypical Mendelian single gene disorders affecting the β-globin gene have the most impact on morbidity and mortality, involving millions of people worldwide (Weatherall, 2010). β-thalassemia defects result in an imbalance accumulation of α- and β-globin proteins inside red blood cells (RBCs). This condition causes red cell membrane damage, early cell death, and ineffective erythropoiesis. SCD is due to a mutation that outcomes in a substitution of the sixth amino acid of adult β-globin. Hemoglobin tetramers bearing this mutation (HbS) polymerize inside RBCs and distort them; this rigid sickle RBCs can block blood vessels in the microcirculation compromising oxygen delivery to tissues. The current routines therapies, besides transfusion and iron chelation, include the treatment with Hydroxyurea (HU), the only fetal hemoglobin (HbF) inducer approved by the U.S. Food and Drug Administration (FDA). Despite this, treatments with HU generate sufficient levels of HbF in only half of patients (Steinberg et al., 1997) and side effects including leukopenia and neutropenia are frequently reported. Therefore, novel therapeutic inducers must be identified in order to develop a personalized treatment of patients with β-thalassemia and sickle cell anemia. Severe clinical complications of β-thalassemia and SCD may occur due to the accumulation of free α-globins or to the production of defective β-globin, respectively. Therefore, we investigated also the reduction of hemoglobin as a potential pathway to target for developing new therapies. Part I of this PhD thesis focuses on the characterization of novel fetal hemoglobin inducers: 1) by collecting 33 blood samples from different patients with β-thalassemia, we have demonstrated the action of Rapamycin to induce fetal hemoglobin production, even in HU-resistant cells; 2) during the in vivo administration to β-thalassemia patients of a Resveratrol-containing nutraceutical we have analyzed the expression of γ-globin mRNA and the production of fetal hemoglobin in erythroid precursors cells; 3) we have tested new psoralens analogues by evaluating the erythroid differentiation of K562 cells and the effects on globin genes expression and HbF production in erythroid precursors cells. Part II is concerned about the potential therapeutic application of peptide nucleic acids (PNAs) in erythroid cells: 1) in human erythroleukemia cells we obtained an efficient liposomemediated delivery of PNA with low antiproliferative activity (Avitabile et al. 2015); 2) an antisense PNA targeting β-globin mRNA was found to inhibit hemoglobin production in murine erythroleukemia cells (Montagner et al., 2015); 3) a PNA-anti-β-glob-SCA was tested in erythroid precursors cells isolated from SCD patients.

Mutations in the beta-globin (β-globin) gene cause beta-thalassaemia (β-thalassaemia).The screening strategy for β-thalassaemiais based on the value of mean corpuscular volume (MCV) from the complete blood count (CBC) data. Current laboratory practice considers blood samples with MCV higher than 80fL as normal. No further assessment will be done on these samples. However, there are clinically silent β-globin gene mutations with MCV higher than 80fL, for example, heterozygous haemoglobin E (HbE). The importance of finding out this kind of mutations is due to the serious outcome when they occur together with classic β thalassaemia mutations in compound heterozygous states, which may produce a condition mimicking β thalassaemia major. The method used to recognize the presence of clinically silent β-globin gene mutations should be robust and with high sensitivity. High resolution melting (HRM) is a suitable technique to screen gene mutations. It is fast and convenient. The process is completed in a closed system without any post PCR manipulation. The sensitivity is up to a single nucleotide change. Using HRM for mutations screening followed by confirmation with sequencing can reduce time and cost of testing clinically silent β-globin gene mutations on a large scale. This study first shows the ability of HRM in detecting various types of β-globin gene mutations. The technique is then applied to detect clinically silent β-globin gene mutations in a group of high school students with normal CBC data. Mutations with different clinically significance were found. The frequency of mutation found in the samples of the study suggests that screening for β-globin gene mutation may be worthwhile in subjects with MCV higher than 80fL.
published_or_final_version
Pathology
Master
Master of Medical Sciences

The thalassaemias are the commonest monogenic disorders in the world population. They occur at a particularly high frequency in Mediterranean regions and Southeast Asia, which cause a massive public health problem. In Hong Kong, the prevalence of heterozygous carriers of α or β thalassaemia mutations is approximately 8% [1]. Thalassaemia is characterized by the reduced synthesis of one or more normal globin chains. This causes globin chain imbalance and finally leads to hypochromic microcytic anaemia. Different types of thalassaemia are named according to the under-produced chains. The majority of thalassaemia can be diagnosed by basic haematologic profiles and simple phenotypic techniques. However, in some cases of thalassaemia the diagnosis are not apparent after routine laboratory investigations. To arrive at a diagnosis which is important for antenatal diagnosis and genetic counseling, it is necessary to use molecular approaches. In this study, 25 patients with microcytosis, normal phenotypic haemoglobin study results and without iron deficiency were analyzed retrospectively. This cohort of patients was suspected to have occult or masked thalassaemia. DNA was extracted from archive samples and further investigated by alpha multiplex gap polymerase chain reaction (α multiplex gap-PCR), alpha amplification refractory mutation system (α ARMS) and direct nucleotide sequencing of globin genes for the detection of possible underlying globin gene mutations. Results indicated that 60% of these cases with microcytosis were occult and silent αthalassaemia caused by deletional or non-deletional mutations. Maskedβthalassaemia due to co-existing δ thalassaemia or variants or normal Hb A2 β thalassaemia due to mild β globin gene mutations were not detected in the cohort. Forty percent of these cases of microcytosis remained unexplained, which await further molecular testing.
published_or_final_version
Pathology
Master
Master of Medical Sciences

Mutations in the ATRX gene are responsible for the alpha-thalassemia mental retardation (ATR-X) syndrome. ATRX is a putative global transcription regulator and chromatin remodelling protein. The goal of this research is to characterize interactions ATRX has with other proteins involved in transcription regulation, and identify domains in ATRX that may be responsible for these interactions. Several stable NIH 3T3 tet-off cell lines have been established that contain a human ATRX transgene. In addition, ATRX, PML, and Daxx appear to co-localize in nuclear bundles, suggesting they may act together transiently, or in a complex, in a regulatory role. A domain has been identified on ATRX that appears to target the protein to nuclear bundles, and interact with PML and Daxx. ATRX patient mutations appear to alter these interactions. This work attempts to elucidate cellular functions of ATRX, in hopes of establishing a better understanding of the neuropathology of this complex disease.

Thalassemia is a genetic disorder that is characterized by abnormal hemoglobin within red blood cells of the body. If not diagnosed early and treated with proper care, this disorder is fatal. Once considered rare, this disorder’s rise in prevalence is now of concern to many health professionals because it does not receive the same level of attention as other, more common blood disorders. Due to its chronic treatment course and recent advancements in treatment, thalassemia still remains primarily a pediatric disorder not having many cases of patients living into adulthood at this point in time [1]. Seeing this shortcoming in thalassemia advocacy we find it useful to take certain steps to bring more public awareness of it. We hope to share information about this debilitating disorder, its management/ treatment course and most importantly, its prevention through genetic testing. We conducted a patient quality improvement study at the Pediatric Hematology/Oncology/BMT clinic at Banner University Medical Center Tucson, AZ to assess the effectiveness of consultations and supplemental education on the treatment and care of thalassemia patients. The focus of the project revolved around patients with the most prominent thalassemias, beta and alpha.